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J Bacteriol, June 1998, p. 3187-3196, Vol. 180, No. 12
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Characterization of the hcnABC Gene Cluster Encoding Hydrogen Cyanide Synthase and Anaerobic Regulation by ANR in the Strictly Aerobic Biocontrol Agent Pseudomonas fluorescens CHA0

Jacques Laville,¹ Caroline Blumer,² Christine Von Schroetter,^1, Valeria Gaia,^1, Geneviève Défago,³ Christoph Keel,² and Dieter Haas^2,*

Mikrobiologisches Institut¹ and Institut für Pflanzenwissenschaften/Phytopathologie, Eidgenössische Technische Hochschule, CH-8092 Zürich,³ and Laboratoire de Biologie Microbienne, Université de Lausanne, CH-1015 Lausanne,² Switzerland

Received 12 December 1997/Accepted 31 March 1998

The secondary metabolite hydrogen cyanide (HCN) is produced by Pseudomonas fluorescens from glycine, essentially under microaerophilic conditions. The genetic basis of HCN synthesis in P. fluorescens CHA0 was investigated. The contiguous structural genes hcnABC encoding HCN synthase were expressed from the T7 promoter in Escherichia coli, resulting in HCN production in this bacterium. Analysis of the nucleotide sequence of the hcnABC genes showed that each HCN synthase subunit was similar to known enzymes involved in hydrogen transfer, i.e., to formate dehydrogenase (for HcnA) or amino acid oxidases (for HcnB and HcnC). These similarities and the presence of flavin adenine dinucleotide- or NAD(P)-binding motifs in HcnB and HcnC suggest that HCN synthase may act as a dehydrogenase in the reaction leading from glycine to HCN and CO₂. The hcnA promoter was mapped by primer extension; the 40 sequence (TTGGC ... .ATCAA) resembled the consensus FNR (fumarate and nitrate reductase regulator) binding sequence (TTGAT ... .ATCAA). The gene encoding the FNR-like protein ANR (anaerobic regulator) was cloned from P. fluorescens CHA0 and sequenced. ANR of strain CHA0 was most similar to ANR of P. aeruginosa and CydR of Azotobacter vinelandii. An anr mutant of P. fluorescens (CHA21) produced little HCN and was unable to express an hcnA-lacZ translational fusion, whereas in wild-type strain CHA0, microaerophilic conditions strongly favored the expression of the hcnA-lacZ fusion. Mutant CHA21 as well as an hcn deletion mutant were impaired in their capacity to suppress black root rot of tobacco, a disease caused by Thielaviopsis basicola, under gnotobiotic conditions. This effect was most pronounced in water-saturated artificial soil, where the anr mutant had lost about 30% of disease suppression ability, compared with wild-type strain CHA0. These results show that the anaerobic regulator ANR is required for cyanide synthesis in the strictly aerobic strain CHA0 and suggest that ANR-mediated cyanogenesis contributes to the suppression of black root rot.

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^* Corresponding author. Mailing address: Laboratoire de Biologie Microbienne, Université de Lausanne, CH-1015 Lausanne, Switzerland. Phone: 41 21 692 56 31. Fax: 41 21 692 56 35. E-mail: Dieter.Haas{at}lbm.unil.ch.

Present address: Institut für Molekularbiologie und Biophysik, ETH, CH-8093 Zürich, Switzerland.

Present address: Istituto Cantonale Batteriosierologico, CH-6904 Lugano, Switzerland.

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J Bacteriol, June 1998, p. 3187-3196, Vol. 180, No. 12
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

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